1. Field of the Invention
The present invention relates to a shipping medium for organ-derived living cells, such as cells from the heart, liver, kidney, pancreas, skin and cornea. More specifically, the invention is directed to a semi-solid cell shipping medium, and method for using said medium, that maximizes the viability and protection of organ-derived living cells during temporary storage and transportation.
2. Background Art
Significant medical advances have been made using organ-derived cells as in vitro models of in vivo systems. There are two distinct types of organ-derived cell preparations that are used for research purposes: primary isolated cells and cell lines. Primary isolated cells are cells that are harvested from living organs and are used within hours to a few days of cell harvest. Cell lines, on the other hand, are typically considered as producing continuous cells since they can be subcultured numerous times before the cells die.
A major advantage of using primary isolated cells rather than cell lines is that primary isolated cells provide characteristic biochemical processes that can be observed in vivo. Unfortunately, primary isolated cells from visceral organs such as the heart, liver, and kidney are relatively fragile and have a viability limited to a few days, unless these cells are transformed into cell lines or are cryopreserved. Primary isolated cells that are transformed into cell lines exhibit a proliferative tendency, causing them to more closely resemble cancerous cells than normal cells.
Cryopreservation of organ-derived cells involves a tedious titration of the cells with a cryoprotectant such as dimethylsulfoxide (DMSO), while the temperature is gradually lowered, until an equilibrium is reached across the plasma membrane. The recovery of cells from cryopreservation involves the reverse process followed by immediate removal of DMSO. Unfortunately, recovery of cells from cryopreservation can result in very low cell yields in the range of twenty-five percent to fifty percent.
Therefore, it is desirable to have a preservation medium and.backslash.or method to preserve the viability of primary isolated cells in such a manner that the cells closely model the in vivo system, but that minimizes the technical expertise and time required to obtain and preserve such cells.
In addition, it is desirable to have a preservation medium and.backslash.or method that not only preserves cells, but allows the efficient banking and shipping of these cells to investigators. The shipping of primary isolated cells of organs, such as cells from the liver, kidney, and heart, remains problematic without cryopreservation when using conventional techniques and storage conditions.
In this regard, the shipment of cells in a preservation medium is fundamentally different from cultivating cells in a conventional growth medium. In the latter case, a nutrient medium containing various cell growth compounds is traditionally used to maintain cells in the same condition as when such cells are in vivo and to encourage life processes, growth and cell proliferation. The continuation of these activities, of course, causes the cells to expend a tremendous amount of energy. The growth medium is usually maintained at a temperature range of 30.degree.-40.degree. C., which is the optimal temperature range for most types of body-derived cells. The growth substrate is usually in a liquid state.
In U.S. Pat. No. 5,336,614 (Soft Agar Assay and Kit), Brown and Schwartz describe an agar-based cell growth formulation that contains a variety of cell growth compounds and that forms a soft gel between 33.degree.-40.degree. C., thereby allowing eukaryotic cells to slowly settle into the gel during culture. Another example of a soft semi-solid growth substrate is Matrigel.TM., a commercially available growth medium that contains various growth compounds. Matrigel.TM. is a "reverse-gelling" compound. In other words, it remains fluid at a low temperature, but becomes a soft gel at higher, growth temperatures. A large amount of research has been conducted recently in the use of Extracellular Matrix (ECM) Overlay in which soft gel growth mediums are used to form "sandwiches" by placing a layer of the soft gel both above and below the cells to be cultured to simulate an in vivo environment and to enhance the biochemical activity of the cells. See, e.g., E. Leycluse, et al. (1994) Am. J. Physiol. C1764-C1774; J. S. Sidhu et al., 1994 In Vitro Toxicology (Vol. 4, No. 3) pg. 225, 229; H. G Koebel et al., (1994) Xenobiotica (Vol. 24, No. 2) pg. 95-107; R. Ezzell et al., (1993), Experimental Cell Research 208, 442-452.
In contrast to a growth medium, a shipping medium must be maintained at relatively low temperatures and should be specifically formulated to arrest or suspend cell replication and cellular biochemical processes, thereby conserving energy and cellular integrity.
A fundamental distinction also exists between the preservation and shipment of organ-derived cells and the preservation and shipment of other cells, such as blood cells. The paramount concern in preserving and/or shipping organ-derived cells, such as liver cells, is to preserve the biochemical function of such cells. These cells are biochemically very active, and tend to degrade quickly in a conventional shipping medium, even at low temperatures. In contrast, the preservation and shipment of platelet cells or other blood cells is fairly simple, because these types of cells have no significant biochemical activity.
There have been a number of formulations used for the preservation of blood cells such as red blood cells, leukocytes, and platelet cells. For example, in U.S. Pat. No. 4,476,221 (Protective Solution for Preserving Functional Cells), Kane et al., disclose a red corpuscle preservation solution. In U.S. Pat. No. 4,004,975 (Method of Isolation and Cryopreserving Human White Cells from Whole Blood), Lionetti et al. describe a method to cryopreserve blood-derived cells. Tullis, in U.S. Pat. No. 2,786,014 (Platelet Preservation), describes a method to store blood platelet in a sterile soft gelatinous matrix that can be directly injected into humans.
As described above, such preservation formulations are not applicable to the preservation and shipment of organ-derived cells, because of the fundamental differences between organ-derived cells and blood cells.
Some organ-derived cells can be shipped at ambient temperature to above-freezing temperature without cryoprotectant in sealed flasks filled with nutrient medium. However, to minimize agitation that could result in cell injury, the sealed flasks require a large excess of nutrient medium to reduce the airspace in the culture vessel. The use of excess liquid medium, however, does not eliminate potentially harmful movement of the cells, is wasteful, and also increases the weight of the shipment, thereby increasing the shipping costs of the sample.
Additionally, a significant percentage of cell research is conducted in cell culture plates or cell culture wells that cannot be easily sealed. Hence, spillage of the liquid medium results if the container is inverted during shipment. Thus, present technology is lacking in the existence of a medium for shipping cells that maintains cellular viability at ambient to above-freezing conditions, that prevents spillage, that protects the cells being transported and that can be easily used by researchers.
In attempting to overcome some of the problems with preserving organ-derived cells, a variety of preservation formulations and/or methods have been developed. For example, several preservation solutions for the storage of organ tissue intended for transplant have been developed. At present, the most widely used cold organ preservation solution for donor organs is VIASPAN.TM., disclosed in Belzer et al., U.S. Pat. No. 4,879,283 (Solution for the Preservation of Organs).
Other organ or organ-derived cell preservation solutions and/or methods that have proven useful have been described in the following patents:
Belzer et al., U.S. Pat. No. 4,798,824 (Perfusate for the Preservation of Organs); and Belzer et al., U.S. Pat. No. 4,873,230 (Composition for the Preservation of Organs), (disclosing hydroxyethyl starch as a composition useful in organ preservation solutions); PA0 Andrews, U.S. Pat. No. 5,306,711 (Organ Preservative Solution) (disclosing an organ preservative containing dextran); PA0 Kazumasa et al., U.S. Pat. No. 4,186,253 (Perfusate for Preserving Organ to be Transplanted and Preserving Method) (describing an organ preservation solution that contains a perfluorocarbon compound to increase the oxygen-carrying capacity of an aqueous buffer); PA0 Hurley, et al., U.S. Pat. No. 5,256,571 (Cell Preservative Solution) (describing an acidified alcohol based fixative to preserve the cellular structure of dead cells); PA0 Brockbank, et al., U.S. Pat. No. 5,110,722 (Cell, Tissue or Organ Storage Solution) (describing a method of maintaining cellular viability at about 4.degree. C. or less in a non-frozen state using a physiological buffer containing selenium and transferrin); PA0 Fahy, et al., U.S. Pat. No. 5,217,860 (Method for Preserving Organs for Transplantation by Vitrification); and U.S. Pat. No. 4,559,298 (Cryopreservation of Biological Materials in a Non-frozen or Vitreous State) (describing methods of cellular preservation by using vitrifiable concentrations of a cryoprotectant); PA0 Swartz, U.S. Pat. No. 4,681,839 (Systems to Preserve Living Tissue) (describing a system to preserve living tissue that has been severed from its host or cells to be stored using a gas permeable bag and a soluble biscuit composed of nutrients); and PA0 Jost, U.S. Pat. No. 4,473,552 (Anaerobic Method for Preserving Whole Blood, Tissue, and Components Containing Living Mammalian Cells) (describing a system to preserve living tissue in an oxygen free sealed receptacle).
Also of interest is Lemasters and Thurman, U.S. Pat. No. 5,145,771 (Rinse Solution for Organs and Tissues), in which the inventors describe how lethal organ injury occurs after stored organs are perfused with warm physiological buffers and disclose a rinse solution that can be used to flush organs immediately prior to transplant. Specific formula components disclosed in Lemasters et al. are: hydroxyethyl starch (similar to the disclosure in U.S. Pat. Nos. 4,873,230, 4,879,283 and 4,798,824) and adenosine (also disclosed in U.S. Pat. Nos. 4,798,824, 4,920,044, and 5,200,398).
The protective effect of mild acidosis (pH between 6.0 to 7.1) is also described in Lemasters et al. as well as by numerous investigators: Bing, O. H., W. W. Brooks, and J. V. Messer (1973) Science 180, 1297-1298; Pentilla, A. and B. F. Trump (1974) Science 185, 277-278; Bonventre, J. V. and J. Y. Cheung (1985) Am. J. Physiol. 249, C149-C159; and Gores, G. J., K. E. Fleishman, T. E. Dawson, B. Herman, A, L. Nieminen, and J. J. Lemasters (1988) Am. J. Physiol. 255, C315-C322; Koop, A. and H. M. Piper (1992) J. Mol. Cell. Cardiol., 24, 55-65.
Systems customized for the preservation of corneal tissue are described in U.S. Pat. No. 4,873,186 (Cornea Storage Medium), U.S. Pat. No. 5,104,787 (Method for Apparatus for a Defined Serum Free Medical Solution Useful for Corneal Preservation), and U.S. Pat. No. 5,166,048 (Protection of Human Corneal Endothelial Cells).
Although each of the above-described formulations and/or methods are successful in addressing some of the problems associated with the preservation of organ-derived cells, none of these formulations and/or methods are formulated for the shipment of such cells and, hence, they do not solve the significant problems associated with shipping organ-derived cells, such as the problems of rapid cell degradation, physical injury to the cells and spillage.
Thus, no present formulation or method for shipping organ-derived cells exists that adequately arrests biochemical activity and preserves cellular integrity for up to several days, that provides physical protection to organ-derived cells during shipment, that eliminates spillage during shipment and that allows transportation of isolated living cells in a ready-to-use culture apparatus, such as multi-well plates, culture flasks or cover slips.